Synthesis of some new quinazolinone derivatives and evaluation of their antimicrobial activities.

WIDE RANGE OF QUINAZOLINONE BIOLOGICAL PROPERTIES INCLUDING
antibacterial, anticancer, and anti-inflammatory activities encouraged us to synthesis some fused quinazolinone derivatives. Anthranilic acid was condensed with chloro acylchloride followed by dehydration to form the benzoxazinone intermediate; subsequent addition of an amine provided the fused quinazolinones. Deoxyvasicinone which was previously synthesized by a multi step complex reactions was prepared in three steps using the following procedure: Log P values of the compounds were measured using the shake flask method in octanol/water solvent system. The synthesized compounds were evaluated against six strains of bacteria (three Gram-positive and three Gram-negative) and three strains of fungi. Overall results of antimicrobial tests showed that the compounds had better bacteriostatic activity against Gram-negative bacteria. The obtained results of MBC revealed that these compounds had more significant bacteriostatic than bactericidal activities. Almost all of the screened compounds showed good activity against C. albicans and A. niger. The obtained results of MFC indicated that these compounds had more significant fungistatic than fungicidal activities.

A highly employed method for 4(3H)quinazolinone synthesis is based on acylation of anthranilic acid with acyl chloride. Subsequent ring closure with acetic anhydride afford corresponding 1,3-benzoxazin-4one (benzoxazinone) which will be treated with different amines to give 4(3H)quinazolinone derivatives (16). Here a number of fused quinazolinone derivatives including deoxyvasicinone were synthesized using this procedure. In this method chloro acyl chloride was used instead of acyl chloride. The second ring closure was achieved simultaneously to produce fused quinazolinone derivatives in acceptable yields. Synthesized compounds were evaluated against six strains of bacteria (three Gram-positive and three Gram-negative) and three strains of fungi.

Chemistry
All solvents and the chemicals used in this study were purchased from Merck Co. (Merck, Germany). Melting points were determined in open capillaries using electrothermal 9200 melting point apparatus and are uncorrected. 1 H-NMR spectra were recorded on a Bruker 400 MHz spectrometer and the chemical shifts are expressed as δ (ppm) with tetramethylsilane as internal standard. Mass spectra were recorded on Shimadzu Mass spectrometer. IR spectra were recorded with a WQF -510 FT-IR spectrophotometer.
The benzoxazinone 8 (0.15 mole) and excess of hydrazine-hydrate were dissolved in ethanol and refluxed for 2 h. The mixture was purified by column chromatography on silica gel using an eluent of CHCl 3 -MeOH 49:1 to give compounds 9 and 10.

CH=C-N).
Synthesis of 2-Benzyl-isoindoline-1, 3-Dione (13) Compound 12 (0.005 mole) and benzyl amine (0.005 mole) refluxed in toluene for 5 h.The precipitated product was collected by filtration. Ethylene glycol and NaOH (0.01 g) were added to residue and heated in an oil bath for 2h.The crystals were filtered off to provide compound 13 as brown crystals (42% )

Log P measurement
Log p measurement is a useful parameter to understand the lipophilicity of drug molecules. The shake flask method is the usual way for measuring log p values. The UV absorbance of an aqueous solution of a compound is measured before and after being shaken together with a known volume of octanol. One advantage of the method is that the appearance of compound in the octanol may be checked against the disappearance from the aqueous phase. It is very important to pre-saturate the solvents in prolonged shake-flask experiments (17, 18).

Determination of partition coefficients using the shake flask method
Partition coefficients (K part ) of the compounds were determined using the shake flask method. The two phases used in determination were tris buffer (50 mM, pH 7.4, prepared using distilled water) and 1-octanol, each of which was preequilibrated with the other phase before use (the solubility of water in 1-octanol is 2.3 M) (17, 18) . The synthesized compounds were dissolved in tris buffer to obtain (10 -4 M) stock solutions. The relationship between absorbance and concentration of samples (10 -5 , 2×10 -5 , 4 ×10 -5 , 6×10 -5 , 8×10 -5) were found linear according to

Synthesis of 2-(4-oxo-4H-benzo [d] [1,3] oxazin-2-yl)-benzoic acid (12)
Anthranilic acid 1 (0.04 mole) was treated with phetalic anhydride (0.04 mole) in glacial acetic acid for 5 h. After completion of the reaction, solvent was distilled off under reduced pressure, the residue was recrystallized from acetone to obtain compound 12 as white crystals (48%)  A known volume (normally 10-50 ml) sample of the solution was stirred vigorously with a suitable volume of 1-octanol in a glass vessel for 1 h. The two layers were separated by centrifugation for 5 min. An aliquot of the aqueous layers was then carefully removed using a glass Pasteur pipette ensuring that the sample was not contaminated with 1-octanol. The absorbance of the sample was measured as above and the partition coefficient was then calculated using the following formula: A 1 = Absorbance reading in the aqueous layer before partitioning. A 2 = Absorbance reading in the aqueous layer after partitioning. V W = Volume of aqueous layer used in partitioning.
V O = Volume of 1-octanol layer used in partitioning. For each compound, the experiment was repeated at least three times for the calculation of a mean K part value and standard deviation. The results are shown in Table 1. Sabouraud dextrose agar was used to culture fungal strains and Mueller Hinton agar was used to culture bacterial strains. The inocula of microorganisms (1.5 × 10 8 CFU/mL) were prepared from cultures and suspensions were adjusted to 0.5 Mc Farland standard turbidity. Synthesized compounds were dissolved in DMSO (0.5 mL) and diluted with water up to 1 mL to obtain concentration of 5120 μg/mL as stock solutions. The serial dilution method was used to obtain 2560 to 320 μg/mL concentrations (19,20).

Antimicrobial activity
Mueller-Hinton broth was used as medium for bacterial growth. 20 µL of each concentration were distributed in 96-well plates with the exception of those wells acting as growth control (contain microorganisms and culture media) and positive control (contain microorganisms and standard antibiotic). After adding Alamar Blue ® reagent (20 µL) to all of the 96 wells total volume in each well became 200 µL. The final concentrations of compounds were (512-32 μg/ mL) and the final concentrations of inocula were 1.5 × 10 4 for bacteria and 1.5 × 10 5 for fungi. Plates were covered and sealed with parafilm and incubated for 24 h at 37°C. The MIC was defined as the lowest concentration, which prevented a color change from blue to pink (20, 21). Ciprofloxacin was used as standard antibacterial drug. The same method except for some modifications was used for the antifungal studies. The incubation time was 48 h at 25°C for fungi. Ketoconazole was used as standard antifungal agent. RPMI 1640 medium was used as medium for fungi (20).
Following a broth microdilution MIC test, from each well that shows no growth, contents were removed and spreaded onto mueller Hinton agar plates for bacteria and sabouraud dextrose agar for fungi to determine MBC and MFC results. The plates were incubated for 24 h at 37°C for bacteria and 25°C for fungi (20).

Results and Discussion
The synthetic pathways to the intermediates and final compounds (2-10) are presented in Figures 1 and 2. Briefly anthranilic acid was condensed with chloro acylchloride to produce N-acyl antranilic acid. First ring closure and  subsequent dehydration were performed with acetic anhydride to form the benzoxazinone intermediate. Finally addition of hydrazine hydrate or ammonium acetate provided the fused quinazolinones (4, 5 and 10). Addition of aniline instead of simple amines resulted in production of compound 11 as presented in Figure 3. In production of compound 4, hydrazinehydrate acts as a nucleophile and attacks the carbonyl group of cyclic ester due to its alpha effect. Simultaneous nucleophilic attacks of carbohydrazine nitrogens to the carbonyl group of amide and methylen chloride end group of the side chain resulted in the production of the tricyclic product 4.
In preparation of deoxyvasicinone 5, ammonia acts as a nucleophile and attacks to the carbonyl group of the cyclic ester. Nucleophilic attacks of amine to carbonyl group of amide and methylene chloride end group of the side chain afforded the second tricyclic compound 5 (Figure 1).
The benzoxazinone 8 and excess of hydrazine-hydrate were reacted in ethanol and the reaction mixture was fractionated by column chromatography. Compound 9 is a result of nucleophilic attack of the solvent (ethanol) instead of NH 2 to the methylene chloride end group of the side chain ( Figure 2). Compound 10 which is a five membered ring analogue of 4 was produced by similar mechanism explained in the production of compound 4.
In an attempt for the production of a quinazolinone derivatives substituted at position 2 and 3 with benzoic acid and benzyl amine, respectively, surprisingly compound 13 ( Figure  4) was obtained. The probable mechanism for production of this unexpected compound 13 is shown in Figure 4.
In conclusion application of chloroacyl chlorides instead of acyl chlorides resulted in a third ring closure. This third ring closure was achieved when an intramolecular nucleophilic attack to the end methylene chloride group was possible. Formation of highly stable five or six membered ring may be counted as a good reason for this ring closure.
Deoxyvasicinone which was previously synthesized by multi step complex reactions (11-15) with long reaction times, harsh reaction conditions or application of microwave irradiation, was easily produced here by  application of bi-functional group starting material, «chloroacyl chloride» instead of usual acyl chlorides.
Obtained results of log p indicated that the presence of chlorine atom at the end of side chain increased lipophilicity of compounds. In fused pyrazol or pyridazine-quinazolinones, incorporation of NH in hetrocyclic ring reduced liphophilicity of molecules (Table1).
Obtained results of tested compounds against Gram positive bacteria showed that compounds 4, 5 and 10 had the highest activities against B. subtitles at 32 or 64 µg/mL concentrations. Results against Gram negative bacteria showed that some compounds showed acceptable activity against P. aeruginosa. Existence of a phenyl group at position 3 of quinazolinone and chlorine atom at the end of side chaine in compound 11 could improve activity against P. aeruginosa in comparison with compound 9. Anthranilic acid derivatives 2 and 7 with a free carboxylic acid and chlorine atom at the end of side chain showed good activity against P. aeruginosa at 32 µg/mL concentration. Compounds 3 with benzoxazinone structure and chlorine atom at the end of the side chain also exhibited activity against P. aeruginosa at 32 µg/mL concentration. This could be due to the unstability of benzoxazinone ring and possible cleavage to free carboxylic acid derivative. Overall results of antibacterial tests revealed that compounds exhibited better bacteriostatic activity against Gram-negative bacteria. The obtained results of MBC revealed that tested compounds have more significant bacteriostatic than bactericidal activities. Almost all of the screened compounds showed good activity against C. albicans and A. niger. While Compounds 4 and 9 showed better activities against both fungi at 32 and 64 µg/mL respectively. The obtained results of MFC revealed that tested compounds have more significant fungistatic than fungicidal activities (Tables 2 and 3). (10)